This invention relates to the preparation of extracts from the blood cells of organisms such as Limulus polyphemus, the horseshoe crab. In particular this invention relates to an improved process for eliminating the variation in performance among various lots of Limulus amebocyte lysates in endotoxin assays.
Coagulase is a proteolytic enzyme, the activity of which is modulated by endotoxin, i.e., the enzyme is allosterically activated by endotoxin. It is not proteolytically active in the absence of endotoxin. Coagulase is known to be found in, and is obtained from the blood amebocytes of Limulus polyphemus and Tachypleus tridentatus.
Coagulase is usually isolated from the amebocytes by lysing the cells to free the water soluble intracellular components and centrifuged to remove suspended detritus. A suitable technique for obtaining such extracts from Limulus is disclosed in United Kingdom Pat. No. 1,499,846. Such cell-free amebocyte isolates are referred to herein as amebocyte extracts.
The amebocyte extracts are known to contain coagulase and coagulogen. Coagulogen is a protein which is hydrolyzed by activated coagulase to form protein fragments. These fragments are thought to then spontaneously polymerize to yield a clot or gel.
Both coagulase and coagulogen have been extensively studied and characterized. Regarding coagulase, consult Tai et al., "Journal of Biological Chemistry" 252(14): 4773-4776 (1977) and Solum, "Thrombosis Research" 2:55-70 (1973). Coagulogen, and a method for its purification, are described by Gaffin in "Biorheology" 13:273-280 (1976).
The high endotoxin sensitivity of coagulase forms the basis for screening assays for endotoxin pyrogens in human body fluids and biological products. Such assays have been particularly useful in the quality control of pharmaceuticals and parenteral solutions. Several conventional methods exist in which endotoxin is assayed by coagulase-mediated clotting of coagulogen. Included among them are those in which the increase in optical density or viscosity of amebocyte lysate is followed upon mixture with samples thought to contain endotoxin, or in which coagulase activity is assayed by measuring the degree of hydrolysis of a synthetic, chromogenic polypeptide substrate by coagulase.
The widespread adoption of such assays on a commercial scale has been hindered by such unreliable performance, particularly variations in apparent sensitivity to endotoxin encountered among separate lots of Limulus extracts. Such variations have been postulated to be based on seasonal factors, i.e., dependent upon the time of year the Limulus blood is collected. Another theory has been that a component is present in the extracts which affect performance. The component has been characterized as an "inhibitor" of the clotting reaction, reflecting the effect obtained in the presence of the inhibitor. It has been speculated that the inhibitor may be an enzyme [Nachum et al., "Journal of Invertebrate Pathology" 32:51-58 (1978)] or a lipoprotein that simply binds endotoxin [Sullivan et al., "Applied Microbiology" 28(6):1023-1026 (1974)]. Whatever its mode of action, the material will hereinafter be designated as an inhibitor.
It has been suggested to reduce inhibitor activity by combining equal volumes of organic solvent and Limulus lysate, shaking, centrifuging and recovering the aqueous phase (Sullivan et al., op cit). Applicants consider this method to be unsatisfactory because it requires handling toxic or highly flammable reagents and calls for a burdensome phase separation. Furthermore, residual organic solvent may remain in the Sullivan et al. product. Even water-immiscible solvents exhibit a slight solubility in water; in the case of chloroform, this solubility is 0.82 parts per 100 parts of water at 20.degree. C. The effect of this residue on the sample to be assayed may be unpredictable or undesirable.
The presence of the inhibitor in extracts which had been considered "standardized" has prevented the extracts from in fact performing as reliable standards. A standardized extract must produce the same experimental outcome from lot to lot, with only insignificant variation. This is impossible if variable activity of interfering substances in the extracts have not been accounted for. Further, the known "standardized" extracts have not been prepared with suitable consideration of coagulogen concentration. Typical "standardized" Limulus lysates are disclosed in U.S. Pat. Nos. 3,944,391 and 4,038,147. These products have been prepared by simply assaying a lysate batch against a serial dilution of endotoxin to determine the gross potency of the lysate. The principal difficulty with this approach is that it is essentially passive; the extract is in no way treated or processed to yield a single predetermined signal, e.g., development of a given nephelometric endpoint upon reaction with a given endotoxin concentration. This renders quality control more difficult since new standard curves must be prepared at the user level for each new lot of lysate. It is commercially desirable to be able to supply lysate from a variety of lots which will continuously produce the same results in an endotoxin determination using constant endotoxin in concentrations even though the original, untreated lots would have produced a scattering of signals, some higher and some lower than the desired, predetermined level.
Thus it is an object of this invention to produce endotoxin clottable extracts which will yield a reproducible predetermined analytical signal for a given endotoxin concentration within an analytically significant range.
It is an additional object of this invention to replace the previously employed inhibitor inactivation process with one which does not require toxic or hazardous reagents, which is less expensive in both labor and materials and which leaves no potentially interfering residue in the product.
These and other objects of this invention will be apparent from a consideration of this specification as a whole.